The Applicants are aware of a number of different techniques and methods used in the separation and purification of blood cell populations (and sub-populations). One of the most commonly used techniques for separating the white blood cells (leucocytes) from the red blood cells (the erythrocytes) is to simply mix a sample of blood with a solution which aggregates the red blood cells to thereby increase their rate of sedimentation. The density of the separating fluid is such that the sedimentation of the white blood cells is only partially affected and can be collected from the upper part of the separation fluid when the red blood cells have sedimented.
A more recently developed technique makes use of a system where the red blood cell aggregating agent is not actually mixed with the blood but rather the blood sample is carefully layered onto the top of the separating fluid medium whereupon the red blood cells are caused to agglutinate or aggregate at the interface and sediment to the bottom of the tube in which the medium is placed. There are several well known high polymer compounds which agglutinate the red blood cells, for example FICOLL 400 (registered Trade Mark of Pharmacia Fine Chemicals, Sweden) which is a neutral highly branched, high molecular weight polymer of sucrose, and these are generally mixed with a compound, in solution, of relatively high density and relatively low viscosity, for example sodium metrizoate or sodium diatrizoate. The separation can be carried out at unit gravity or by centrifugation. The majority of the white blood cells remain at the interface but such previous systems have not been effective in fractionating the white blood cell subpopulations, namely the mononuclear and the polymophonuclear cells--at least not in a one-step process using a separating medium of single density. In order to achieve such separation, one known method involves the isolation of the mononuclear white blood cells by centrifugation as a first step using a Isopaque-Ficoll (registered Trade Mark) mixture (Isopaque solution being obtained from Nyegaard & Co. of Norway and having as its main component sodium metrizoate) followed by separation of the polymorphonuclear white blood cells by using dextran or gelatin in order to sediment the red blood cells. Other known methods have used dextran sedimentation as a first step to obtain mixed white blood cells (leucocytes), followed by a second step of centrifugation using a Isopaque-Ficoll density gradient medium in order to separate the white blood cell sub-populations. A further step is required to lyse contaminating red blood cells with ammonium chloride in order to obtain relatively pure polymorphonuclear cells. It is also known to make use of a discontinuous density gradient where 2 or more separating solutions of different densities are layered on top of one another. The densities are selected so as to provide a (discontinuous) gradient over the desired range.
In another reported development, a one-step process was used but was only effective in separating the mononuclear leucocytes from the whole blood sample. For example, it was known to use a separation medium consisting of a mixture of Hypaque-Ficoll having a density of 1.077 g/ml onto which the blood sample was layered. Following centrifugation of the blood, the red blood cells and the polymorphonuclear leucocytes sedimented to the bottom while the mononuclear leucocytes formed a band at the interface. The separated fraction of mononuclear white blood cells was separately recovered by pipetting off the interface layer. The principles of density separation of the blood cell populations by the centrifugation method is well known to those skilled in the art and hence need not be discussed in any detail.